Methods of using improved polymerases

ABSTRACT

This invention provides for methods of sequencing and performing polymerase reactions using an improved generation of nucleic acid polymerases. The improvement is the fusion of a sequence-non-specific nucleic-acid-binding domain to the enzyme in a manner that enhances the processivity of the polymerase.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/306,827 filed Nov. 27, 2001, which claims the benefit of U.S.provisional application No. 60/333,966 filed Nov. 28, 2001, both ofwhich are incorporated by reference herein.

FIELD OF THE INVENTION

This invention provides more efficient methods of performing polymerasereactions. The methods employ an improved generation of nucleic acidpolymerases. The improvement is the joining sequence-non-specificnucleic-acid-binding domain to the enzyme in a manner that enhances theability of the enzyme to bind and catalytically modify the nucleic acid.

BACKGROUND OF THE INVENTION

The processivity of a polymerase, i.e., the amount of product generatedby the enzyme per binding event, can be enhanced by increasing thestability of the modifying enzyme/nucleic acid complex. The currentinvention now provides enhanced polymerase assays that employ novelmodifying enzymes in which the double-stranded conformation of thenucleic acid is stabilized and the processivity of the enzyme increasedby joining a sequence-non-specific double-stranded nucleic acid bindingdomain to the enzyme, or its catalytic domain which are disclosed e.g.,in co-pending U.S. application Ser. No. 09/870,353 and WO01/92501. Themodifying proteins that are processive in nature exhibit increasedprocessivity when joined to a binding domain compared to the enzymealone.

There is a need to enhance polymerase reactions in many applications.For example, SYBR Green I (Molecular Probes, Eugene, Oreg.; U.S. Pat.Nos. 5,436,134 and 5,658,751), a fluorescent dye that is specific fordsDNA detection, is widely used in real-time PCR reactions to monitorthe generation of dsDNA through each cycle of amplification. However,the addition of SYBR Green I inhibits the activity of DNA polymerasesused in PCR. Similarly, it is often desirable to use PCR for theanalysis of crude or “dirty” nucleic acid samples. For example, colonyPCR is a useful technique in which small samples of single bacterialcolonies are lysed and added directly to PCR reactions for the purposeof screening colonies for particular DNA sequences. However, colony PCRhas a high failure rate, because of residual contaminants from thecolony. Thus, polymerases that are resistant to such inhibitors, e.g.,fluorescent dyes and impurities present in the cell extracts, are neededin order to obtain more efficient polymerase reactions, e.g., PCR.

There is also a need to improve sequencing reactions. Polymerasescurrently employed in sequencing reactions, e.g., cycle sequencing, areoften inefficient. For example, cycle sequencing is often performed withpoorly-processive enzymes. Often, the enzymes used are ΔTaq derivatives,which have Taq polymerase's 5′-3′ nuclease domain removed, and have aprocessivity of about 2 bases. Also, in the case of dyeterminator-sequencing, dITP is used in place of dGTP, which causespolymerase pausing and dissociation at G nucleotides. These enzymestherefore produce a large number of sequence products that areimproperly terminated. These stops compete with, and negatively effect,the production of properly terminated sequence products. Furthermore, ifa polymerase dissociates during primer extension of a templatecontaining a repeat unit (e.g., a triplet repeat) or secondary structure(e.g., a stem and loop), the 3′ end can denature and reanneal so as toprime at a different location on the template—for example, in the caseof a repeat, the reannealing could occur at a different repeat; or inthe case of secondary structure, improper reannealing could delete out asection of the template. Thus, dissociation of the polymerase duringsequencing can cause a problem in efficiently obtaining reliablesequencing information.

The current invention addresses both of these needs, i.e., the need forenhancing polymerase reactions performed in the presence of inhibitorsand the need for enhancing processivity in DNA sequencing applications).The current invention provides such enhanced, or improved, polymerasereactions. The improvement is the use of a polymerase that has increasedprocessivity due to the presence of a sequence-non-specificnucleic-acid-binding domain that is joined to the polymerase.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of performing more efficientpolymerase reactions using a polymerase protein comprising a polymerasedomain joined to a sequence-non-specific double-stranded nucleic acidbinding domain. Typically the presence of the sequence non-specificdouble-stranded nucleic acid binding domain enhances the processivity ofthe polymerase compared to an identical protein not having asequence-non-specific nucleic acid binding domain joined thereto.

The polymerase domain can be thermally stable, e.g., a Thermuspolymerase domain such as a ΔTaq polymerase domain, or a Pyrococcuspolymerase domain.

In one embodiment the sequence-non-specific nucleic-acid-binding domainspecifically binds to polyclonal antibodies generated against eitherSac7d or Sso7d. Alternatively, the sequence-non-specificnucleic-acid-binding domain contains a 50 amino acid subsequencecontaining 50% amino acid similarity to Sso7d. Typically, thesequence-non-specific nucleic-acid-binding domain is Sso7d orspecifically binds to polyclonal antibodies generated against Sso7d.

The polymerase reaction can be performed on a target nucleic acid thatis present in a crude preparation of a sample. In another embodiment,the polymerase reaction is performed in the presence of a molecule thattypically inhibits polymerases, e.g. fluorescent dyes such as SYBR GreenI. Further, the polymerase may be used in cycle sequencing reactions toobtain longer sequences, e.g., through regions of secondary structurethat prevent sequencing using unmodified polymerases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the results of a PCR reaction performed in thepresence of contaminants using an improved polymerase.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Archaeal small basic DNA-binding protein” refers to protein of between50-75 amino acids having either 50% homology to a natural Archaeal smallbasic DNA-binding protein such as Sso-7d from Sulfolobus sulfataricus orbinds to antibodies generated against a native Archaeal small basicDNA-binding protein.

“Domain” refers to a unit of a protein or protein complex, comprising apolypeptide subsequence, a complete polypeptide sequence, or a pluralityof polypeptide sequences where that unit has a defined function. Thefunction is understood to be broadly defined and can be ligand binding,catalytic activity or can have a stabilizing effect on the structure ofthe protein.

“Efficiency” in the context of a nucleic acid modifying enzyme of thisinvention refers to the ability of the enzyme to perform its catalyticfunction under specific reaction conditions. Typically, “efficiency” asdefined herein is indicated by the amount of product generated undergiven reaction conditions.

“Enhances” in the context of an enzyme refers to improving the activityof the enzyme, i.e., increasing the amount of product per unit enzymeper unit time.

“Fused” refers to linkage by covalent bonding.

“Heterologous”, when used with reference to portions of a protein,indicates that the protein comprises two or more domains that are notfound in the same relationship to each other in nature. Such a protein,e.g., a fusion protein, contains two or more domains from unrelatedproteins arranged to make a new functional protein.

“Join” refers to any method known in the art for functionally connectingprotein domains, including without limitation recombinant fusion with orwithout intervening domains, intein-mediated fusion, non-covalentassociation, and covalent bonding, including disulfide bonding; hydrogenbonding; electrostatic bonding; and conformational bonding, e.g.,antibody-antigen, and biotin-avidin associations.

“Nucleic-acid-modifying enzyme” refers to an enzyme that covalentlyalters a nucleic acid.

“Polymerase” refers to an enzyme that performs template-directedsynthesis of polynucleotides. The term, as used herein, also refers to adomain of the polymerase that has catalytic activity.

“Error-correcting activity” of a polymerase or polymerase domain refersto the 3′ to 5′ exonuclease proofreading activity of a template-specificnucleic acid polymerase whereby nucleotides that do not formWatson-Crick base pairs with the template are removed from the 3′ end ofan oligonucleotide, i.e., a strand being synthesized from a template, ina sequential manner. Examples of polymerases that have error-correctingactivity include polymerases from Pryococcus furiosus, Thermococcuslitoralis, and Thermotoga maritima.

Processivity refers to the ability of a nucleic acid modifying enzyme toremain bound to the template or substrate and perform multiplemodification reactions. Processivity is measured by the number ofcatalytic events that take place per binding event.

“Sequence-non-specific nucleic-acid-binding domain” refers to a proteindomain which binds with significant affinity to a nucleic acid, forwhich there is no known nucleic acid which binds to the protein domainwith more than 100-fold more affinity than another nucleic acid with thesame nucleotide composition but a different nucleotide sequence.

“Thermally stable polymerase” as used herein refers to any enzyme thatcatalyzes polynucleotide synthesis by addition of nucleotide units to anucleotide chain using DNA or RNA as a template and has an optimalactivity at a temperature above 45° C.

“Thermus polymerase” refers to a family A DNA polymerase isolated fromany Thermus species, including without limitation Thermus aquaticus,Thermus brockianus, and Thermus thermophilus; any recombinant enzymesderiving from Thermus species, and any functional derivatives thereof,whether derived by genetic modification or chemical modification orother methods known in the art.

The term “amplification reaction” refers to any in vitro means formultiplying the copies of a target sequence of nucleic acid. Suchmethods include but are not limited to polymerase chain reaction (PCR),DNA ligase reaction (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCRProtocols: A Guide to Methods and Applications (Innis et al., eds,1990)), (LCR), QBeta RNA replicase, and RNA transcription-based (such asTAS and 3SR) amplification reactions as well as others known to those ofskill in the art.

“Amplifying” refers to a step of submitting a solution to conditionssufficient to allow for amplification of a polynucleotide if all of thecomponents of the reaction are intact. Components of an amplificationreaction include, e.g., primers, a polynucleotide template, polymerase,nucleotides, and the like. The term “amplifying” typically refers to an“exponential” increase in target nucleic acid. However, “amplifying” asused herein can also refer to linear increases in the numbers of aselect target sequence of nucleic acid.

The term “amplification reaction mixture” refers to an aqueous solutioncomprising the various reagents used to amplify a target nucleic acid.These include enzymes, aqueous buffers, salts, amplification primers,target nucleic acid, and nucleoside triphosphates. Depending upon thecontext, the mixture can be either a complete or incompleteamplification reaction mixture

“Polymerase chain reaction” or “PCR” refers to a method whereby aspecific segment or subsequence of a target double-stranded DNA, isamplified in a geometric progression. PCR is well known to those ofskill in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; andPCR Protocols: A Guide to Methods and Applications, Innis et al., eds,1990. Exemplary PCR reaction conditions typically comprise either two orthree step cycles. Two step cycles have a denaturation step followed bya hybridization/elongation step. Three step cycles comprise adenaturation step followed by a hybridization step followed by aseparate elongation step.

“Long PCR” refers to the amplification of a DNA fragment of 5 kb orlonger in length. Long PCR is typically performed usingspecially-adapted polymerases or polymerase mixtures (see, e.g., U.S.Pat. Nos. 5,436,149 and 5,512,462) that are distinct from thepolymerases conventionally used to amplify shorter products.

A “primer” refers to a polynucleotide sequence that hybridizes to asequence on a target nucleic acid and serves as a point of initiation ofnucleic acid synthesis. Primers can be of a variety of lengths and areoften less than 50 nucleotides in length, for example 12-30 nucleotides,in length. The length and sequences of primers for use in PCR can bedesigned based on principles known to those of skill in the art, see,e.g., Innis et al., supra.

A temperature profile refers to the temperature and lengths of time ofthe denaturation, annealing and/or extension steps of a PCR or cyclesequencing reaction. A temperature profile for a PCR or cycle sequencingreaction typically consists of 10 to 60 repetitions of similar oridentical shorter temperature profiles; each of these shorter profilesmay typically define a two step or three-step cycle. Selection of atemperature profile is based on various considerations known to those ofskill in the art, see, e.g., Innis et al., supra. In a long PCR reactionas described herein, the extension time required to obtain anamplification product of 5 kb or greater in length is reduced comparedto conventional polymerase mixtures.

PCR “sensitivity” refers to the ability to amplify a target nucleic acidthat is present in low copy number. “Low copy number” refers to 10⁵,often 10⁴, 10³, 10², or fewer, copies of the target sequence in thenucleic acid sample to be amplified.

A “template” refers to a double stranded polynucleotide sequence thatcomprises the polynucleotide to be amplified, flanked by primerhybridization sites. Thus, a “target template” comprises the targetpolynucleotide sequence flanked by hybridization sites for a 5′ primerand a 3′ primer.

An “improved polymerase” includes a sequence-non-specificdouble-stranded DNA binding domain joined to the polymerase orpolymerase domain. An “unimproved polymerase” is a polymerase that doesnot have a sequence-non-specific double-stranded DNA binding domain.

Introduction

The current invention provides methods of performing polymerasereactions using improved polymerases. These polymerase reactions aretypically more efficient and yield more product than traditionalpolymerases. These improved polymerases contain a polymerase domain witha binding domain joined to it. While the prior art taught that nucleicacid binding proteins can increase the binding affinity of enzymes tonucleic acid, the group of binding proteins having the ability toenhance the processive nature of the enzymes is of particular value. Notto be bound by theory, binding domains of the invention typicallydissociate from double-stranded nucleic acid at a very slow rate. Thus,they increase the processivity and/or efficiency of a modifying enzymeto which they are joined by stabilizing the enzyme-nucleic acid complex.Accordingly, this invention results from the discovery that DNA-bindingdomains can stabilize the double-stranded conformation of a nucleic acidand increase the efficiency of a catalytic domain that requires adouble-stranded substrate. Described herein are examples and simpleassays to readily determine the improvement to the catalytic and/orprocessive nature of catalytic nucleic acid modifying enzymes, e.g.,polymerases.

Polymerase Domains.

DNA polymerases are well-known to those skilled in the art. Theseinclude both DNA-dependent polymerases and RNA-dependent polymerasessuch as reverse transcriptase. At least five families of DNA-dependentDNA polymerases are known, although most fall into families A, B and C.There is little or no structural or sequence similarity among thevarious families. Most family A polymerases are single chain proteinsthat can contain multiple enzymatic functions including polymerase, 3′to 5′ exonuclease activity and 5′ to 3′ exonuclease activity. Family Bpolymerases typically have a single catalytic domain with polymerase and3′ to 5′ exonuclease activity, as well as accessory factors. Family Cpolymerases are typically multi-subunit proteins with polymerizing and3′ to 5′ exonuclease activity. In E. coli, three types of DNApolymerases have been found, DNA polymerases I (family A), II (familyB), and III (family C). In eukaryotic cells, three different family Bpolymerases, DNA polymerases α, δ, and ε, are implicated in nuclearreplication, and a family A polymerase, polymerase γ, is used formitochondrial DNA replication. Other types of DNA polymerases includephage polymerases.

Similarly, RNA polymerases typically include eukaryotic RNA polymerasesI, II, and III, and bacterial RNA polymerases as well as phage and viralpolymerases. RNA polymerases can be DNA-dependent and RNA-dependent.

In one embodiment, polymerase domains that have an error-correctingactivity are used as the catalytic domain of the improved polymerasesdescribed herein. These polymerases can be used to obtain long, i.e., 5kb, often 10 kb, or greater in length, PCR products. “Long PCR” usingthese improved polymerases can be performed using extension times thatare reduced compared to prior art “long PCR” polymerase and/orpolymerase mixtures. Extension times of less than 30 seconds per kb,often 15 seconds per kb, can be used to amplify long products in PCRreactions using the improved polymerases. Furthermore, these modifiedpolymerases also exhibit increased sensitivity.

Prior-art non-error-correcting polymerases such as Taq polymerase arecapable of amplifying DNA from very small input copy concentrations,such as, in the extreme, 10 copies per ml. However, because of the lowfidelity of such polymerases, products cloned from such amplificationsare likely to contain introduced mutations.

Prior-art error-correcting polymerases such as Pfu copy DNA with higherfidelity than Taq, but are not capable of amplifying DNA from smallinput copy concentrations. The hybrid error-correcting polymerases ofthe invention exhibit much higher processivity while retainingerror-correcting activity and thereby provide both sensitivity andfidelity in amplification reactions.

The activity of a polymerase can be measured using assays well known tothose of skill in the art. For example, a processive enzymatic activity,such as a polymerase activity, can be measured by determining the amountof nucleic acid synthesized in a reaction, such as a polymerase chainreaction. In determining the relative efficiency of the enzyme, theamount of product obtained with a polymerase containing asequence-non-specific double-stranded DNA binding domain can then becompared to the amount of product obtained with the normal polymeraseenzyme, which will be described in more detail below and in theExamples.

A polymerase domain suitable for use in the invention can be the enzymeitself or the catalytic domain, e.g., Taq polymerase or a domain of Taqwith polymerase activity. The catalytic domain may include additionalamino acids and/or may be a variant that contains amino acidsubstitutions, deletions or additions, but still retains enzymaticactivity.

Sequence-Non-Specific Nucleic-Acid-Binding Domain

A double-stranded sequence-non-specific nucleic acid binding domain is aprotein or defined region of a protein that binds to double-strandednucleic acid in a sequence-independent manner, i.e., binding does notexhibit a gross preference for a particular sequence. Typically,double-stranded nucleic acid binding proteins exhibit a 10-fold orhigher affinity for double-stranded versus single-stranded nucleicacids. The double-stranded nucleic acid binding proteins in particularembodiments of the invention are preferably thermostable. Examples ofsuch proteins include, but are not limited to, the Archaeal small basicDNA binding proteins Sac7d and Sso7d (see, e.g., Choli et al.,Biochimica et Biophysica Acta 950:193-203, 1988; Baumann et al.,Structural Biol. 1:808-819, 1994; and Gao et al, Nature Struc. Biol.5:782-786, 1998), Archael HMf-like proteins (see, e.g., Starich et al.,J. Molec. Biol. 255:187-203, 1996; Sandman et al., Gene 150:207-208,1994), and PCNA homologs (see, e.g., Cann et al., J. Bacteriology181:6591-6599, 1999; Shamoo and Steitz, Cell: 99, 155-166, 1999; DeFelice et al., J. Molec. Biol. 291, 47-57, 1999; and Zhang et al.,Biochemistry 34:10703-10712, 1995).

Sso7d and Sac7d

Sso7d and Sac7d are small (about 7,000 kd MW), basic chromosomalproteins from the hyperthermophilic archaeabacteria Sulfolobussolfataricus and S. acidocaldarius, respectively. These proteins arelysine-rich and have high thermal, acid and chemical stability. Theybind DNA in a sequence-independent manner and when bound, increase theT_(M) of DNA by up to 40° C. under some conditions (McAfee et al.,Biochemistry 34:10063-10077, 1995). These proteins and their homologsare typically believed to be involved in packaging genomic DNA andstabilizing genomic DNA at elevated temperatures.

HMF-Like Proteins

The HMf-like proteins are archaeal histones that share homology both inamino acid sequences and in structure with eukaryotic H4 histones, whichare thought to interact directly with DNA. The HMf family of proteinsform stable dimers in solution, and several HMf homologs have beenidentified from thermostable species (e.g., Methanothermus fervidus andPyrococcus strain GB-3a). The HMf family of proteins, once joined to TaqDNA polymerase or any DNA modifying enzyme with a low intrinsicprocessivity, can enhance the ability of the enzyme to slide along theDNA substrate and thus increase its processivity. For example, thedimeric HMf-like protein can be covalently linked to the N terminus ofTaq DNA polymerase, e.g., via chemical modification, and thus improvethe processivity of the polymerase.

PCNA Homologs

Many but not all family B DNA polymerases interact with accessoryproteins to achieve highly processive DNA synthesis. A particularlyimportant class of accessory proteins is referred to as the slidingclamp. Several characterized sliding clamps exist as trimers insolution, and can form a ring-like structure with a central passagecapable of accommodating double-stranded DNA. The sliding clamp formsspecific interactions with the amino acids located at the C terminus ofparticular DNA polymerases, and tethers those polymerases to the DNAtemplate during replication. The sliding clamp in eukarya is referred toas the proliferating cell nuclear antigen (PCNA), while similar proteinsin other domains are often referred to as PCNA homologs. These homologshave marked structural similarity but limited sequence similarity.

Recently, PCNA homologs have been identified from thermophilic Archaea(e.g., Sulfalobus sofataricus, Pyroccocus furiosus, etc.). Some family Bpolymerases in Archaea have a C terminus containing a consensusPCNA-interacting amino acid sequence and are capable of using a PCNAhomolog as a processivity factor (see, e.g., Cann et al., J. Bacteriol.181:6591-6599, 1999 and De Felice et al., J. Mol. Biol. 291:47-57,1999). These PCNA homologs are useful sequence-non-specificdouble-stranded DNA binding domains for the invention. For example, aconsensus PCNA-interacting sequence can be joined to a polymerase thatdoes not naturally interact with a PCNA homolog, thereby allowing a PCNAhomolog to serve as a processivity factor for the polymerase. By way ofillustration, the PCNA-interacting sequence from Pyrococcus furiosusPolII (a heterodimeric DNA polymerase containing two family B-likepolypeptides) can be covalently joined to Pyrococcus furiosus PolI (amonomeric family B polymerase that does not normally interact with aPCNA homolog). The resulting fusion protein can then be allowed toassociate non-covalently with the Pyrococcus furiosus PCNA homolog togenerate a novel heterologous protein with increased processivityrelative to the unmodified Pyrococcus furiosus PolI.

Other Sequence-Nonspecific Double-Stranded Nucleic Acid Binding Domains

Additional nucleic acid binding domains suitable for use in theinvention can be identified by homology with known sequence non-specificdouble-stranded DNA binding proteins and/or by antibody crossreactivity,or may be found by means of a biochemical assay. These methods aredescribed, e.g., in WO01/92501. Further, methods of joining thepolymerase to the sequence non-specific double-stranded DNA bindingprotein and methods of expressing recombinant polymerases and polymerasefusion proteins are also described (see, e.g., WO01/92501).

Assays to Determine Improved Activity of Polymerase Domains.

Activity of the polymerase domain can be measured using a variety ofassays that can be used to compare processivity or modification activityof a modifying protein domain joined to a binding domain compared to theprotein by itself. Improvement in activity includes both increasedprocessivity and increased efficiency.

Polymerase processivity can be measured in variety of methods known tothose of ordinary skill in the art. Polymerase processivity is generallydefined as the number of nucleotides incorporated during a singlebinding event of a modifying enzyme to a primed template.

For example, a 5′ FAM-labeled primer is annealed to circular orlinearized ssM13mp18 DNA to form a primed template. In measuringprocessivity, the primed template usually is present in significantmolar excess to the enzyme or catalytic domain to be assayed so that thechance of any primed template being extended more than once by thepolymerase is minimized. The primed template is therefore mixed with thepolymerase catalytic domain to be assayed at a ratio such asapproximately 4000:1 (primed DNA:DNA polymerase) in the presence ofbuffer and dNTPs. MgCl₂ is added to initiate DNA synthesis. Samples arequenched at various times after initiation, and analyzed on a sequencinggel. At a polymerase concentration where the median product length doesnot change with time or polymerase concentration, the length correspondsto the processivity of the enzyme. The processivity of a protein of theinvention, i.e., a protein that contains a sequence non-specificdouble-stranded nucleic acid binding domain fused to the catalyticdomain of a processive nucleic acid modifying enzyme such as apolymerase, is then compared to the processivity of the enzyme withoutthe binding domain.

Enhanced efficiency can also be demonstrated by measuring the increasedability of an enzyme to produce product. Such an analysis measures thestability of the double-stranded nucleic acid duplex indirectly bydetermining the amount of product obtained in a reaction. For example, aPCR assay can be used to measure the amount of PCR product obtained witha short, e.g., 12 nucleotide in length, primer annealed at an elevatedtemperature, e.g., 50° C. In this analysis, enhanced efficiency is shownby the ability of a polymerase such as a Taq polymerase to produce moreproduct in a PCR reaction using the 12 nucleotide primer annealed at 50°C. when it is joined to a sequence-non-specific double-strandednucleic-acid-binding domain of the invention, e.g., Sso7d, than Taqpolymerase does alone. In contrast, a binding tract that is a series ofcharged residues, e.g. lysines, does not enhance processivity whenjoined to a polymerase.

Assays such as salt sensitivity can also be used to demonstrateimprovement in efficiency of a processive nucleic acid modifying enzymeof the invention. A modifying enzyme, or the catalytic domain, whenfused to a sequence non-specific double-stranded nucleic acid bindingdomain of the invention exhibits increased tolerance to high saltconcentrations, i.e., a processive enzyme with increased processivitycan produce more product in higher salt concentrations. For example, aPCR analysis can be performed to determine the amount of productobtained in a reaction using a fusion Taq polymerase (e.g., Sso7d fusedto Taq polymerase) compared to Taq polymerase in reaction conditionswith high salt, e.g., 80 mM.

Other methods of assessing enhanced efficiency of the improvedpolymerases of the invention can be determined by those of ordinaryskill in the art using standard assays of the enzymatic activity of agiven modification enzyme.

Uses of Improved Polymerases

The invention provides improved methods of performing polymerasereactions. In one embodiment, the invention provides a method ofperforming a polymerase reaction in the presence of a fluorescent dye. Anumber of fluorescent dyes that are commonly used in reactions such asreal-time PCR, have an inhibitory activity on polymerases have beentypically used in PCR, e.g., Taq polymerase. For example, SYBR Green I(Molecular Probes, Eugene, Oreg.; U.S. Pat. Nos. 5,436,134 and5,658,751), is a fluorescent dye that is specific for dsDNA detection,and is widely used in real-time PCR reactions to monitor the generationof dsDNA through each cycle of amplification. Use of dyes to monitoramplification is described in U.S. Pat. Nos. 5,994,056 and 6,171,785 anduse of SYBR Green I for this purpose is described in Morrison et al.,Biotechniques 24:954-962 (1998).

It has been observed that the addition of SYBR Green I inhibits theactivity of DNA polymerases used in PCR, possibly through interferingwith the binding of the polymerase to the primer-template. Additivessuch as DMSO are therefore often required to reduce the inhibitoryeffect of the dye. However, DMSO can reduce the storage stability of theenzyme and can inhibit polymerases. The current invention provides amethod of performing polymerase reactions in the presence of afluorescent dye that uses the improved polymerases described herein,which are not as sensitive to the fluorescent dye, i.e., are notinhibited to the same extent, as an unimproved polymerase.

The ability of a polymerase to perform in the presence of a dye thatexhibits altered fluorescence emissions when bound to double-strandedDNA can be measured using well known polymerase assays, such as thosedescribed herein. Typically, a fluorescent dye reduces the activity ofan unimproved polymerase by 25%, often 50%, 75%, or more. Polymeraseactivity can be assayed using the methods described herein.

The ability of an improved polymerase to perform a PCR reaction in thepresence of a fluorescent dye, e.g., SYBR Green I, can also be comparedto the ability of the unimproved polymerase to perform in an otherwiseidentical PCR reaction. The comparison can be made using a values suchas the cycle threshold (C_(t)) value, which represents the number ofcycles required to generate a detectable amount of DNA. An efficientpolymerase may be able to produce a detectable amount of DNA in asmaller number of cycles by more closely approaching the theoreticalmaximum amplification efficiency of PCR. Accordingly, a lower C_(t)value reflects a greater amplification efficiency for the enzyme. Theimproved enzymes exhibit 2×, often 5×, or greater activity in thepresence of a fluorescent dye when compared to the unimproved enzyme.

In typical embodiments, the polymerase reaction is performed in thepresence of a fluorescent dye such as SYBR Green I or Pico Green I(Molecular Probes, Eugene, Oreg.;). These dyes are unsymmetrical cyaninedyes containing a defined substituent on the pyridinium or quinoliniumring system or a substituent immediately adjacent to the nitrogen atomof the pyridinium or quinolinium ring. These and other members of thesame class of dyes are described, e.g., in U.S. Pat. Nos. 5,436,134 and5,658,751. SYBR Green I, for example, binds specifically to dsDNA with adissociation constant in the sub-micromolar range. Upon binding, it hasa large increase in its quantum yield and therefore a large increase influorescence.

In other embodiments, the polymerase reactions of the invention can beperformed in the presence of other fluorescent compounds that typicallyinhibit polymerases, such as other fluorescent dyes, e.g., propidiumiodide, ethidium bromide, acridines, proflavine, acridine orange,acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine,distamycin D, chromomycin, mithramycin, ruthenium polypyridyls, andanthramycin, which also exhibit altered fluorescence emissions whenbound to double-stranded DNA. Improved polymerases can be tested forresistance to other dyes using methodology well known in the arts anddescribed herein (see, e.g., Example 6).

In another embodiment, the invention provides method of performing apolymerase reaction, e.g., a PCR reaction, in the presence ofcontaminants such as those present when using a crude nucleic acidsample. Inhibitors of polymerase activity are often present in crudenucleic acid sample preparations, thus presenting difficulties in usingsuch preparations in polymerase reactions such as PCR or nucleic acidsequence. The improved enzymes are more tolerant to such contaminants.Accordingly, the improved enzymes offer advantages over standard enzymeswhen performing polymerase reactions, e.g. PCR, using crude nucleic acidpreparations. These preparations can be from a variety of sources,including cells such as bacterial cells, plant cells, and various othercell types.

A crude nucleic acid sample typically includes contaminants thatoriginate from the nucleic acid source or from a previous chemical ormolecular biological manipulation. The improved polymerases are lesssensitive to the presence of such contaminants. As noted above,polymerase activity assays can be performed using methods describedherein. An improved polymerase typically exhibits 2×, 5×, 10×, orgreater activity relative to the unimproved polymerase when assayed inthe presence of contaminants in an otherwise identical polymeraseactivity assay or PCR. An exemplary analysis of polymerase activity incrude preparations is provided in Example 7. Crude preparationstypically are not processed through repeated rounds of purification andare typically less than 98% pure, often less than 95% pure.

The modified polymerase enzymes are also more resistant to commonadditives for troublesome PCR reactions such as Betaine, DMSO, as wellas resistant to salt, e.g., KCl, etc. The improved polymerase typicallyexhibits 2×, 5×, 10× or greater activity relative to the unimprovedpolymerase in the presence of such agents.

Improved polymerases can also be used in nucleic acid sequencingreactions. These reactions are well known to those of skill in the art(see, e.g., Sambrook and Russell, Molecular Cloning, A Laboratory Manual3rd. 2001, Cold Spring Harbor Laboratory Press).

Improved polymerases are particular advantageous when used in sequencingreactions, in particular sequencing reactions that use thermostablepolymerases, such as cycle sequencing reactions. Cycle sequencing refersto a linear amplification DNA sequencing technique in which a singleprimer is used to generate labeled terminated fragments by the Sangerdideoxy chain termination method. Thermostable polymerase enzymes areemployed in such reactions.

Thermostable polymerases such as Taq or Pfu catalyze the incorporationof ddNTPs at a rate that is at least two orders of magnitude slower thanthe rate of incorporation of dNTPs. In addition, the efficiency ofincorporation of a ddNTP at a particular site is affected by the localsequence of the template DNA. Modified version of polymerases that lack5′ to 3′ exonuclease activity and catalyze incorporation of ddNTPs withhigh efficiency have been developed; however, their processivity isoften poor. For example, thermostable enzymes are such as ΔTaqderivatives, which have Taq polymerase's 5′-3′ nuclease domain removed,have a processivity of about 2 bases. Also, in the case of dyeterminator-sequencing, dITP is used in place of dGTP, which causespolymerase pausing and dissociation at G nucleotides. These enzymestherefore produce a large number of sequence products that areimproperly terminated. Furthermore, if a polymerase dissociates duringprimer extension of a template containing a repeat unit (e.g., a tripletrepeat) or secondary structure (e.g., a stem and loop) such that thestrand is not completed during a particular PCR cycle, the 3′ end candenature and reanneal during a subsequent PCR cycle so as to prime at adifferent location on the template—for example, in the case of a repeat,the reannealing could occur at a different repeat; or in the case ofsecondary structure, improper reannealing could delete out a section ofthe template. Thus, dissociation of the polymerase is also a problem.

The use of improved polymerases as described herein can provide enhancedsequencing reactions, e.g., cycle sequencing reactions, in which thereare fewer improper terminations and fewer dissociation events. Thisprovides longer sequence reads, i.e., the number of nucleotides forwhich the sequence can be determined, that contain fewer ambiguitiescompared to reaction performed with unimproved enzymes.

The polymerases are typically modified to substitute a Y residue for anF residue (U.S. Pat. No. 5,614,365).

All publications, patents, and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill will readily recognize a variety ofnon-critical parameters that could be changed or modified to yieldessentially similar results.

Example 1 Construction of Fusion Proteins

Construction of Sso7d-ΔTaq Fusion

The following example illustrates the construction of a polymeraseprotein possessing enhanced processivity, in which thesequence-non-specific double-stranded nucleic acid binding protein Sso7dis fused to the Thermus aquaticus PolI DNA polymerase (a family Apolymerase known as Taq DNA polymerase) that is deleted at the Nterminus by 289 amino acids (ΔTaq).

Based on the published amino acid sequence of Sso7d, sevenoligonucleotides were used in constructing a synthetic gene encodingSso7d. The oligonucleotides were annealed and ligated using T4 DNAligase. The final ligated product was used as the template in a PCRreaction using two terminal oligonucleotides as primers to amplify thefull-length gene. By design, the resulting PCR fragment contains aunique EcoRI site at the 5′ terminus, and a unique BstXI site at the 3′terminus. In addition to encoding the Sso7d protein, the above PCRfragment also encodes a peptide linker with the amino acid sequence ofGly-Gly-Val-Thr (SEQ ID NO:34) positioned at the C terminus of the Sso7dprotein. The synthetic gene of Sso7d has the DNA sequence shown in SEQID NO:1, and it encodes a polypeptide with the amino acid sequence shownin SEQ ID NO:2.

The synthetic gene encoding Sso7d was then used to generate a fusionprotein in which Sso7d replaces the first 289 amino acid of Taq. Thefragment encoding Sso7d was subcloned into a plasmid encoding Taqpolymerase to generate the fusion protein, as follows. Briefly, the DNAfragment containing the synthetic Sso7d gene was digested withrestriction endonucleases EcoRI and BstXI, and ligated into thecorresponding sites of a plasmid encoding Taq. As the result, the regionthat encodes the first 289 amino acid of Taq is replaced by thesynthetic gene of Sso7d. This plasmid (pYW1) allows the expression of asingle polypeptide containing Sso7d fused to the N terminus of ΔTaq viaa synthetic linker composed of Gly-Gly-Val-Thr (SEQ ID NO:34). The DNAsequence encoding the fusion protein (Sso7d-ΔTaq) and the amino acidsequence of the protein are shown in SEQ ID NOs:3 and 4, respectively.

Construction of Sso7d-Taq Fusion.

An Sso7d/full-length Taq fusion protein was also constructed. Briefly, a1 kb PCR fragment encoding the first 336 amino acids of Taq polymerasewas generated using two primers. The 5′ primer introduces a SpeI siteinto the 5′ terminus of the PCR fragment, and the 3′ primer hybridizesto nucleotides 1008-1026 of the Taq gene. The fragment was digested withSpeI and BstXI, releasing a 0.9 kb fragment encoding the first 289 aminoacids of Taq polymerase. The 0.9 kb fragment was ligated into plasmidpYW1 at the SpeI (located in the region encoding the linker) and BstXIsites. The resulting plasmid (pYW2) allows the expression of a singlepolypeptide containing the Sso7d protein fused to the N terminus of thefull length Taq DNA polymerase via a linker composed of Gly-Gly-Val-Thr(SEQ ID NO:34), the same as in Sso7d-ΔTaq. The DNA sequence encoding theSso7d-Taq fusion protein and the amino acid sequence of the protein areshown in SEQ ID. NOS:5 and 6, respectively.

Construction of Pfu-Sso7d Fusion.

A third fusion protein was created, joining Sso7d to the C terminus ofPyrococcus furiosus DNA poll (a family B DNA polymerase known as Pfu). ApET-based plasmid carrying the Pfu DNA polymerase gene was modified sothat a unique KpnI site and a unique SpeI site are introduced at the 3′end of the Pfu gene before the stop codon. The resulting plasmid (pPFKS)expresses a Pfu polymerase with three additional amino acids(Gly-Thr-His) at its C terminus.

Two primers were used to PCR amplify the synthetic Sso7d gene describedabove to introduce a Kpn I site and a NheI site flanking the Sso7d gene.The 5′ primer also introduced six additional amino acids(Gly-Thr-Gly-Gly-Gly-Gly; SEQ ID NO:35), which serve as a linker, at theN terminus of the Sso7d protein. Upon digestion with KpnI and NheI, thePCR fragment was ligated into pPFKS at the corresponding sites. Theresulting plasmid (pPFS) allows the expression of a single polypeptidecontaining Sso7d protein fused to the C terminus of the Pfu polymerasevia a peptide linker (Gly-Thr-Gly-Gly-Gly-Gly; SEQ ID NO:35). The DNAsequence encoding the fusion protein (Pfu-Sso7d) and the amino acidsequence of the fusion protein are shown in SEQ ID. NOS:7 and 8,respectively.

Construction of Sac7d-ΔTaq Fusion.

A fourth fusion protein was constructed, which joined asequence-non-specific DNA binding protein from a different species toΔTaq. Two primers were used to PCR amplify the Sac7d gene from genomicDNA of Sulfolobus acidocaldarius. The primers introduced a unique EcoRIsite and a unique SpeI site to the PCR fragment at the 5′ and 3′termini, respectively. Upon restriction digestion with EcoRI and SpeI,the PCR fragment was ligated into pYW1 (described above) at thecorresponding sites. The resulting plasmid expresses a singlepolypeptide containing the Sac7d protein fused to the N terminus of ΔTaqvia the same linker as used in Sso7d-ΔTaq. The DNA sequence of thefusion protein (Sac7d-ΔTaq) and the amino acid sequence of the proteinare shown in SEQ ID. NOs: 9 and 10, respectively.

Construction of PL-ΔTaq Fusion

A fifth fusion protein joins a peptide composed of 14 lysines and 2arginines to the N terminus of ΔTaq. To generate the polylysine(PL)-ΔTaq fusion protein, two 67 nt oligonucleotides were annealed toform a duplexed DNA fragment with a 5′ protruding end compatible with anEcoRI site, and a 3′ protruding end compatible with an SpeI site. TheDNA fragment encodes a lysine-rich peptide of the following composition:NSKKKKKKKRKKRKKKGGGVT (SEQ ID NO:36). The numbers of lysines andarginines in this peptide are identical to those in Sso7d. This DNAfragment was ligated into pYW1, predigested with EcoRI and SpeI, toreplace the region encoding Sso7d. The resulting plasmid (pLST)expresses a single polypeptide containing the lysine-rich peptide fusedto the N terminus of ΔTaq. The DNA sequence encoding the fusion protein(PL-ΔTaq) and the amino acid sequence of the protein are shown in SEQID. NOS:11 and 12, respectively.

Example 2 Assessing the Processivity of the Fusion Polymerases

This example illustrates enhancement of processivity of the fusionproteins of the invention generated in Example 1.

Polymerase Unit Definition Assay

The following assay was used to define a polymerase unit. Anoligonucleotide was pre-annealed to ssM13 mp18 DNA in the presence ofMg⁺⁺-free reaction buffer and dNTPs. The DNA polymerase of interest wasadded to the primed DNA mixture. MgCl₂ was added to initiate DNAsynthesis at 72° C. Samples were taken at various time points and addedto TE buffer containing PicoGreen (Molecular Probes, Eugene Oreg.). Theamount of DNA synthesized was quantified using a fluorescence platereader. The unit activity of the DNA polymerase of interest wasdetermined by comparing its initial rate with that of a control DNApolymerase (e.g., a commercial polymerase of known unit concentration).

Processivity Assay

Processivity was measured by determining the number of nucleotidesincorporated during a single binding event of the polymerase to a primedtemplate.

Briefly, 40 nM of a 5′ FAM-labeled primer (34 nt long) was annealed to80 nM of circular or linearized ssM13 mp18 DNA to form the primedtemplate. The primed template was mixed with the DNA polymerase ofinterest at a molar ratio of approximately 4000:1 (primed DNA:DNApolymerase) in the presence of standard PCR buffer (free of Mg⁺⁺) and200 μM of each dNTPs. MgCl₂ was added to a final concentration of 2 mMto initiate DNA synthesis. At various times after initiation, sampleswere quenched with sequencing loading dye containing 99% formamide, andanalyzed on a sequencing gel. The median product length, which isdefined as the product length above or below which there are equalamounts of products, was determined based on integration of alldetectable product peaks. At a polymerase concentration for which themedian product length change with time or polymerase concentration, thelength corresponds to the processivity of the enzyme. The rangespresented in Table 1 represent the range of values obtained in severalrepeats of the assay.

TABLE 1 Comparison of processivity Median product DNA polymerase length(nt) ΔTaq 2-6 Sso7d-ΔTaq 39-58 PL-ΔTaq 2-6 Taq 15-20 Sso7d-Taq 130-160Pfu 2-3 Pfu-Sso7d 35-39

In comparing the processivity of modified enzyme to the unmodifiedenzyme, ΔTaq had a processivity of 2-6 nucleotides, whereas Sso7d-ΔTaqfusion exhibited a processivity of 39-58 nucleotides (Table I). Fulllength Taq had a processivity of 15-20 nucleotides, which wassignificantly lower than that of Sso7d-Taq fusion with a processivity of130-160 nucleotides. These results demonstrate that Sso7d joined to Taqpolymerase enhanced the processivity of the polymerase.

Pfu belongs to family B of polymerases. Unlike Taq polymerase, Pfupossesses a 3′ to 5′ exonuclease activity, allowing it to maintain highfidelity during DNA synthesis. A modified Pfu polymerase, in which Sso7dis fused to the C terminus of the full length Pfu polymerase, and anunmodified Pfu polymerase were analyzed in the processivity assaydescribed above. As shown in Table I, the Pfu polymerase exhibited aprocessivity of 2-3 nt, whereas the Pfu-Sso7d fusion protein had aprocessivity of 35-39 nt. Thus, the fusion of Sso7d to the C terminus ofPfu resulted in a >10-fold enhancement of the processivity over theunmodified enzyme.

Example 3 Effect of Fusion Proteins on Oligonucleotide AnnealingTemperature

This experiment demonstrates the increased efficiency of the Sso7d-ΔTaqfusion protein, compared to Taq, to produce product at higher annealingtemperatures by stabilizing dsDNA.

Two primers, primer 1008 (19mer; T_(M)=56.4° C.) and 2180R (20mer;T_(M)=56.9° C.), were used to amplify a 1 kb fragment (1008-2180) of theTaq pol gene. A gradient thermal cycler (MJ Research, Waltham Mass.) wasused to vary the annealing temperature from 50° C. to 72° C. in a PCRcycling program. The amounts of PCR products generated using identicalnumber of units of Sso7d-ΔTaq and Taq were quantified and compared. Theresults are shown in Table 2. The Sso7d-ΔTaq fusion protein exhibitedsignificantly higher efficiency than full length Taq at higher annealingtemperatures. Thus, the presence of Sso7d in cis increases the meltingtemperature of the primer on the template.

The annealing temperature assay above was used to investigate whetherPL-ΔTaq has any effect on the annealing temperature of primer during PCRamplification. As shown in Table 2 little or no amplified product wasobserved when the annealing temperature was at or above 63° C.

TABLE 2 Comparison of activities at different annealing temperatures.Activity Activity Activity Polymerase at 63° C. at 66° C. at 69° C. Taq  85% 30% <10% Sso7d-ΔTaq >95% 70%   40% PL-ΔTaq  <5% nd nd nd: notdetectable.

Example 4 Effect of Fusion Proteins on Required Primer Length

An enhancement of T_(M) of the primers (as shown above) predicts thatshorter primers could be used by Sso7d-ΔTaq, but not by Taq, to achieveefficient PCR amplification. This analysis shows that Sso7d-ΔTaq is moreefficient in an assay using shorter primers compared to Taq.

Primers of different lengths were used to compare the efficiencies ofPCR amplification by Sso7d-ΔTaq and by Taq. The results are shown inTable 3. When two long primers, 57F (22mer, T_(M)=58° C.) and 732R(24mer, T_(M)=57° C.) were used, no significant difference was observedbetween Sso7d-ΔTaq and Taq at either low or high annealing temperatures.When medium length primers, 57F15 (15mer, T_(M)=35° C.) and 732R16(16mer, T_(m)=35° C.), were used, Sso7d-ΔTaq was more efficient thanTaq, especially when the annealing temperature was high. The moststriking difference between the two enzymes was observed with shortprimers, 57F12 (12mer) and 732R16 (16mer), where Sso7d-ΔTaq generated 10times more products than Taq at both low and high annealingtemperatures.

PCR using primers 57F12 (12 nt) and 732R16 (16 nt) were used to comparethe efficiency of Sac7d-ΔTaq to the unmodified full length Taq in PCRreaction. Similar to Sso7d-ΔTaq, Sac7d-ΔTaq is significantly moreefficient than Taq in amplifying using short primers.

A primer length assay was used to determine the ability of PL-ΔTaq touse short primers in PCR amplification. When long primers (57F and 732R)were used, the amplified product generated by PL-ΔTaq is ˜50% of that bySso7d-ΔTaq. When short primers (57F12 and 732R16) were used, theamplified product generated by PL-ΔTaq is <20% of that by Sso7d-ΔTaq.

TABLE 3 Comparison of the effect of primer length on PCR amplificationby Sso7d-ΔTaq and Taq DNA polymerase. 22 nt primer 15 nt primer 12 ntprimer Anneal Anneal Anneal Anneal Anneal Anneal polymerase @55° C. @63°C. @49° C. @54° C. @49° C. @54° C. Taq 14000 9000 5500 <500 1000undetectable Sso7d-ΔTaq 17000 13000 15000 5000 10000 3000 Sso7d-ΔTaq:Taq1.2:1 1.4:1 2.7:1 >10:1 10:1 >10:1

Increased Performance of Fusion Polymerases in PCR Reactions

The increased stability and/or processivity of the fusion proteins ofthe invention provide increased efficiency in performing variousmodification reactions. For example, polymerase fusion proteins canprovide more efficient amplification in PCR reactions. Many factorsinfluence the outcome of a PCR reaction, including primer specificity,efficiency of the polymerase, quality, quantity and GC-content of thetemplate, length of the amplicon, etc. Examples 5-8 demonstrate thatfusion proteins that include a double-stranded sequence-non-specificnucleic acid binding domain, e.g., Sso7d, joined to a thermostablepolymerase or polymerase domain have several advantageous features overthe unmodified enzyme in PCR applications.

Example 5 Sso7D Fusion Proteins Exhibit a Higher and BroaderSalt-Tolerance in PCR

The binding of polymerase to a primed DNA template is sensitive to theionic strength of the reaction buffer due to electrostatic interactions,which is stronger in low salt concentration and weaker in high. Thepresence of Sso7d in a fusion polymerase protein stabilizes the bindinginteraction of the polymerase to DNA template. This example demonstratesthat Sso7d fusion proteins exhibit improved performance in PCR reactionscontaining elevated KCl concentrations.

Lambda DNA (2 μM) was used as a template in a PCR reactions with primers57F and 732R. The concentration of KCl was varied from 10 mM to 150 mM,while all other components of the reaction buffer were unchanged. ThePCR reaction was carried out using a cycling program of 94° C. for 3min, 20 cycles of 94° C. for 30 sec, 55° C. for 30 sec, and 72° C. for30 sec, followed by 72° C. for 10 min. Upon completion of the reaction,5 μl of the PCR reaction was removed and mixed with 195 μl of 1:400dilution of PicoGreen in TE to quantify the amounts of amplicongenerated. The PCR reaction products were also analyzed in parallel onan agarose gel to verify that amplicons of expected length weregenerated (data not shown). The effects of KCl concentration on the PCRefficiency of Sso7d-ΔTaq versus that of ΔTaq, and Pfu-Sso7d versus Pfuare shown in Table 4. The unmodified enzymes, ΔTaq and Pfu, showed apreference for KCl concentration below 25 mM and 40 mM, respectively, tomaintain 80% of the maximum activity. In contrast, fusion proteinsSso7d-ΔTaq and Pfu-Sso7d maintain 80% of the maximum activity in 30-100mM and 60-100 mM KCl, respectively. Thus, the Sso7d fusion proteins weremore tolerant of elevated KCl concentration in comparison to theirunmodified counter parts. This feature of the hybrid polymerase willpotentially allow PCR amplification from low quality of DNA template,e.g., DNA samples prepared from, but not limited to, blood, food, andplant sources.

TABLE 4 Sso7d modification increases salt-tolerance of polymerase in PCRreaction Enzyme [KCl] for Enzyme concentration 80% activity ΔTaq 20 U/ml  <25 mM Sso7d-ΔTaq 20 U/ml 30-100 mM Pfu  3 U/ml   <40 mM Pfu-Sso7d 12U/ml* (equal molar) 60-100 mM *Pfu-Sso7d has a 4-fold higher specificactivity than Pfu. The specific activity is defined as unit/mol ofenzyme.

Example 6 Sso7d-Fusion Polymerases are More Tolerant to SYBR Green I inReal-Time PCR

Three pairs of unmodified and modified enzymes were compared: commercialΔTaq (ABI, Foster City, Calif.) vs. Sso7d-ΔTaq, Taq vs. Sso7d-Taq, andcommercial Pfu (Stratagene, La Jolla Calif.) vs. Pfu-Sso7d. In additionto the 20 U/ml concentration used for all enzymes, a 5-fold higherconcentration (100 U/ml) of ΔTaq and Pfu were used as well. The Ctvalues represent the number of cycles required to generate a detectableamount of DNA, and thus a lower Ct value reflects a greateramplification efficiency for the enzyme. Consistent Ct values are alsopreferable, indicating the reaction is robust to differences in dyeconcentration. Two extension times (10s and 30s) were used. The SYBRGreen 1 concentration is indicated as 0.5×, etc. The 1×SYBR Green I isdefined as a SYBR Green I solution in TE (10 mM Tris pH 7.5, 1 mM EDTA)that has an absorbance at 495 nm of 0.40±0.02. SYBR Green I waspurchased from Molecular Probes (Eugene, Oreg.) as a 10,000× stock inDMSO. In all three pairs, the modified polymerase showed significantlyhigher tolerance of dye. The differences are most striking in the caseof ΔTaq vs. Sso7d-ΔTaq.

TABLE 5 Sso7d fusion proteins are more tolerant of SYBR Green I. MgCl2 2mM 3 mM SYBR Green I SYBR Green I ENZYMES Unit/ml 0.5x 1x 1.5x 2x 2.5x0.5x 1x 1.5x 2x 2.5x 10 s @ 72° C. ΔTaq 20 — — — — — — — — — — ΔTaq 100— — — — — — — — — — Sso7d-ΔTaq 20 23.3 22.5 22.5 22.3 22.4 22.9 22.2 2222.2 21.8 Taq 20 23 23.6 — — — 22.5 22.3 22.6 — — Sso7d-Taq 20 23.3 23.323.2 23.5 — 24 24 23.1 23.4 23.6 Pfu 20 31.2 — — — — 31.5 — — — — Pfu100 21.8 25 — — — 22.6 23.3 30 — — Pfu-Sso7d 20 21.5 22.3 35 — — 21.8 2222.6 27.2 — 30 s @ 72° C. ΔTaq 20 — — — — — — — — — — ΔTaq 100 — — — — —26.8 — — — — Sso7d-ΔTaq 20 23.8 22.3 22.6 21.8 21.7 22.3 21 21.3 21.821.8 Taq 20 24.2 24.6 29.4 ∞ — 22.8 22.1 22.6 25 — Sso7d-Taq 20 24.223.5 23 22.7 24.2 24.7 23.1 23.6 23.1 22.9 Pfu 20 33.2 — — — — 29.4 — —— — Pfu 100 27.6 30.6 — — — 24.8 29.8 — — — Pfu-Sso7d 20 25 24.8 25.424.4 — 23.1 25.3 23.6 26.1 — (The symbol “—” indicates that noamplification was observed in 40 cycles

Example 7 Sso7d-Fusion Polymerases are More Tolerant to Crude TemplatePreparations A. Resistance to Bacterial Contamination in PCR

Colony PCR is a useful technique in which small samples of singlebacterial colonies are lysed and added directly to PCR reactions for thepurpose of screening the colonies for particular DNA sequences. ColonyPCR has a high failure rate, presumably because of contaminants carriesover from the colony. Polymerases resistant to cell extracts aredesirable because they presumably will be more successful in colony PCR.

Materials For “Dirty” PCR

Lambda template (10 ng/ml): amplicon is a 891 bp fragment

Primers 56F/55R (T_(M) 56° and 55°), 400 nM

Enzymes: Sst (Sso7d-ΔTaq) vs. PE Stf (ΔTaq), STq (Sso7d-Taq) vs. Taq-HISor AmpliTaq or Amersham Taq, and Stratagene Pfu vs. PfS (Pfu-Sso7d) Allenzymes are 20 U/ml except where indicated200 μM each dNTP2 mM MgCl₂, except 1.5 mM for Amersham Taq and AmpliTaqReactions were 20 μl

Methods:

E. coli were grown to saturation, spun down, suspended in water at an ODof 100, and frozen and thawed to disrupt cells. Dilutions of thedisrupted bacteria were added at various concentrations to PCR reactionscontaining lambda DNA as template and two primers to amplify a 890 bpamplicon. 1× is equivalent to an OD of 10 (100D units/ml). The cyclingconditions were as follows:

1) 95° C. −20″ 2) 94° C. −5″ 3) 60° C. −15″ 4) 72° C. −45″

5) repeat steps 2-4 19 times

6) 72° C. −5′

7) 4° C. forever

8) END

The experiment showed that Sso7d-ΔTaq significantly out performedStoffel fragment (Applied Biosystems, Foster City, Calif.). Stoffel(Stf) is a trade name for a preparation of ΔTaq. Using 20 U/ml enzyme inthe final reaction, Sso7d-ΔTaq allowed PCR amplification in the presenceof 0.25× of cell dilution. When the same unit concentrations of Stoffelwas used, no detectable product was generated, even in the most dilutecell solution. When 220 u/ml Stoffel was used, a detectable amount ofproduct was generated at a 0.06× or lower concentration of the celldilution. Thus, the resistance of Sso7d-ΔTaq to bacterial contaminationin PCR reaction is more than 10-folder higher than that of theunmodified enzyme Stoffel.

Similarly, Pfu-Sso7d showed more resistance to bacterial contaminationthan Pfu, although both enzymes appeared to be more sensitive to thecontamination than Taq-based enzymes. With 20 U/ml enzyme in the finalreaction, Pfu allowed amplification only in the presence of 0.00006× orlower concentrations of cell dilution. In contrast, Pfu-Sso7d allowedefficient PCR amplification in 0.002× of cell dilution. Thus, Pfu-Sso7dhas a 30-fold higher tolerance to bacterial contamination in PCR thanthe unmodified enzyme Pfu.

B. Resistance to Plant and Blood Contamination in PCR

The same problems exist with other crude template preparations. PCRfails due to contaminants carried over in the template preparation. Thisexample shows results with crude plant and blood preps. Dilution serieswere made of plant leaf homogenate from Fritallaria agrestis, a speciesof lily, and whole human blood. Dilutions were made with 1×TE, pH 8.0 at1/10, 1/100, 1/1000. One microliter of a dilution was added to theappropriate reaction mix. The PCR cycling protocol was as follows:

94° C. 2 min

94° C. 10 sec

59° C. 20 sec for Taq & Sso7d-Taq (54° C. for Pfu & Pfu-Sso7d)

72° C. 30 sec

repeat cycle 34 times

72° C. 10 min

The reaction products were analyzed on agarose gels (FIG. 1A and FIG.1B). FIG. 1A shows a comparison of the contamination resistance of Pfuvs. PfS. Lanes 1-4 and 14-17 show progressive 10-fold dilutions of plantleaf homogenate. Pfu shows significant inhibition by a 1:10 dilution(lane 2), while PfS is completely resistant to this dilution (lane 7).Similarly, lanes 6-9 and 19-22 show progressive 10-fold dilutions ofblood. Pfu is significantly inhibited by 1 microliter of blood, whilePfS is resistant. Lanes 10 and 23 are positive controls (no plant orblood), while lanes 11 and 24 are negative controls (no plant or bloodor template).

FIG. 1B shows a comparison between Taq and Sso7d-Taq. The upper panelshows reactions performed with 20 U/ml Taq, and the lower panel showsreactions performed with 20 U/ml Sso7d-Taq. Lanes 1-4 in each panel showprogressive 10-fold dilutions of plant leaf homogenate and lanes 7-10show progressive 10-fold dilutions of blood. Sso7d-Taq can amplify aproduct even in the presence of 1 μl whole blood, while Taq is inhibitedby 100-fold less blood. Lanes 5 are positive controls (no plant orblood), while lanes 11 are negative controls (no plant or blood ortemplate).

Example 8 Sso7d-Fusion Polymerases have Advantages in Cycle Sequencing

Plasmid clones encoding improved polymerases suitable for DNA sequencinghave been constructed, and the protein products have been purified. andpurified. The first enzyme is Sso7d-ΔTaq(Y), (SEQ ID No: 30 and 31 withmutations indicated in bold font) which is the same as the enzymeSso7d-ΔTaq, except modified according to the method of Tabor andRichardson (U.S. Pat. No. 5,614,365) to have a “Y” substituted for an“F” residue at the indicated position in SEQ ID NO:31. The second enzymeis Sso7d-ΔTaq(E5;Y) (SEQ ID No: 32 and 33) with mutations indicated inbold font) which is the same as Sso7d-Taq, except modified according tothe method of Tabor and Richardson and also containing point mutationsthat inactivate the 5′-3′ nuclease domain.

The processivity of each Sso7d fusion polymerase was compared to itsunmodified counterpart, i.e., the polymerase without the Sso7d domain.The results in Table 6 show that the Sso7d fusion polymerases are moreprocessive.

TABLE 6 Median Processivity Product Length at 10 mM KCl ΔTaq (Y)  3 to 4nts. Sso7d-ΔTaq (Y) 11 to 13 nts. ΔTaq (E5) (Y)  5 to 6 nts. Sso7d- 34to 47 nts. ΔTaq (E5) (Y)

Sequencing reactions using the fusion polymerases and their unmodifiedcounterparts were performed by separating the components of a commercialsequencing kit (BigDye terminator Kit v.3, ABI, Foster City Calif.).Low-molecular-weight components were separated from the enzymes byultrafiltration. Sequencing reactions performed by combining thelow-molecular-weight fraction with the improved enzymes showed goodsignal strength vs. base number curves. Furthermore, the improvedpolymerases, e.g., Sso7d-ΔTaq(E5;Y), was able to continued through ahard stop better the other enzymes. Such an improved polymerase is alsoable to continue through dinucleotide, trinucletide, and long singlebase repeats more effectively than a counterpart polymerase.

Optimization of the sequencing reactions will demonstrate improvementsin peak height evenness, contamination resistance, and loweredrequirement for template and/or enzyme concentration.

Table of sequences SEQ ID NO: 1 Synthetic Sso7d geneGCAACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGTGGTGCGGTAAGCGAAAAGGACGCGCCGAAGGAGCTGCTGCAGATGCTGGAGAAGCAGAAAAAGSEQ ID NO: 2 The amino acid sequence of Sso7dATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDA PKELLQMLEKQKKSEQ ID NO: 3 The DNA sequence encoding the Sso7d- ΔTaq fusion proteinATGATTACGAATTCGAGCGCAACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGTGGTGCGGTAAGCGAAAAGGACGCGCCGAAGGAGCTGCTGCAGATGCTGGAGAAGCAGAAAAAGGGCGGCGGTGTCACTAGTCCCAAGGCcCTGGAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGTGGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCCCCGAGGGGGTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTGGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCGCCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCCGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAACATCCCCGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCGAGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGATAGAGCTCAGGGTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTCTTCCAGGAGGGGCGGGACATCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGGAGGCCGTGGACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTTCGGGGTCCTCTACGGCATGTCGGCCCACCGCCTCTCCCAGGAGCTAGCCATCCCTTACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAGGCCCGGGTGAAGAGCGTGCGGGAGGCGGCCGAGCGCATGGCCTTCAACATGCCCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGGCTATGGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCCCGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGGGCATTGATGGCCGCGGCGGAGGCGGGCATCATCATCATCATCATTAASEQ ID NO: 4 The amino acid sequence of Sso7d-ΔTaq fusion proteinMITNSSATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQMLEKQKKGGGVTSPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEGIDGRGGGGHHHHHHSEQ ID NO: 5 The DNA sequence encoding the Sso7d- Taq fusion proteinATGATTACGAATTCGAGCGCAACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGTGGTGCGGTAAGCGAAAAGGACGCGCCGAAGGAGCTGCTGCAGATGCTGGAGAAGCAGAAAAAGGGCGGCGGTGTCACTAGTGGGATGCTGCCCCTCTTTGAGCCCAAGGGCCGGGTCCTCCTGGTGGACGGCCACCACCTGGCCTACCGCACCTTCCACGCCCTGAAGGGCCTCACCACCAGCCGGGGGGAGCCGGTGCAGGCGGTCTACGGCTTCGCCAAGAGCCTCCTCAAGGCCCTCAAGGAGGACGGGGACGCGGTGATCGTGGTCTTTGACGCCAAGGCCCCCTCCTTCCGCCACGAGGCCTACGGGGGGTACAAGGCGGGCCGGGCCCCCACGCCAGAGGACTTTCCCCGGCAACTCGCCCTCATCAAGGAGCTGGTGGACCTCCTGGGGCTGGCGCGCCTCGAGGTCCCGGGCTACGAGGCGGACGACGTCCTGGCCAGCCTGGCCAAGAAGGCGGAAAAGGAGGGCTACGAGGTCCGCATCCTCACCGCCGACAAAGACCTTTACCAGCTCCTTTCCGACCGCATCCACGTCCTCCACCCCGAGGGGTACCTCATCACCCCGGCCTGGCTTTGGGAAAAGTACGGCCTGAGGCCCGACCAGTGGGCCGACTACCGGGCCCTGACCGGGGACGAGTCCGACAACCTTCCCGGGGTCAAGGGCATCGGGGAGAAGACGGCGAGGAAGCTTCTGGAGGAGTGGGGGAGCCTGGAAGCCCTCCTCAAGAACCTGGACCGGCTGAAGCCCGCCATCCGGGAGAAGATCCTGGCCCACATGGACGATCTGAAGCTCTCCTGGGACCTGGCCAAGGTGCGCACCGACCTGCCCCTGGAGGTGGACTTCGCCAAAAGGCGGGAGCCCGACCGGGAGAGGCTTAGGGCCTTTCTGGAGAGGCTTGAGTTTGGCAGCCTCCTCCACGAGTTCGGCCTTCTGGAAAGCCCCAAGGCcCTGGAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGTGGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCCCCGAGGGGGTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTGGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCGCCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCCGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAACATCCCCGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCGAGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGATAGAGCTCAGGGTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTCTTCCAGGAGGGGCGGGACATCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGGAGGCCGTGGACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTTCGGGGTCCTCTACGGCATGTCGGCCCACCGCCTCTCCCAGGAGCTAGCCATCCCTTACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAGGCCCGGGTGAAGAGCGTGCGGGAGGCGGCCGAGCGCATGGCCTTCAACATGCCCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGGCTATGGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCCCGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGGGCATTGATGGCCGCGGCGGAGGCGGGCATCATCA TCATCATCATTAASEQ ID NO: 6 The amino acid sequence of Sso7d-Taq fusion proteinMITNSSATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQMLEKQKKGGGVTSGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDW LSAKEGIDGRGGGGHHHHHHSEQ ID NO: 7 The DNA sequence encoding the Pfu- Sso7d fusion proteinATGATTTTAGATGTGGATTACATAACTGAAGAAGGAAAACCTGTTATTAGGCTATTCAAAAAAGAGAACGGAAAATTTAAGATAGAGCATGATAGAACTTTTAGACCATACATTTACGCTCTTCTCAGGGATGATTCAAAGATTGAAGAAGTTAAGAAAATAACGGGGGAAAGGCATGGAAAGATTGTGAGAATTGTTGATGTAGAGAAGGTTGAGAAAAAGTTTCTCGGCAAGCCTATTACCGTGTGGAAACTTTATTTGGAACATCCCCAAGATGTTCCCACTATTAGAGAAAAAGTTAGAGAACATCCAGCAGTTGTGGACATCTTCGAATACGATATTCCATTTGCAAAGAGATACCTCATCGACAAAGGCCTAATACCAATGGAGGGGGAAGAAGAGCTAAAGATTCTTGCCTTCGATATAGAAACCCTCTATCACGAAGGAGAAGAGTTTGGAAAAGGCCCAATTATAATGATTAGTTATGCAGATGAAAATGAAGCAAAGGTGATTACTTGGAAAAACATAGATCTTCCATACGTTGAGGTTGTATCAAGCGAGAGAGAGATGATAAAGAGATTTCTCAGGATTATCAGGGAGAAGGATCCTGACATTATAGTTACTTATAATGGAGACTCATTCGACTTCCCATATTTAGCGAAAAGGGCAGAAAAACTTGGGATTAAATTAACCATTGGAAGAGATGGAAGCGAGCCCAAGATGCAGAGAATAGGCGATATGACGGCTGTAGAAGTCAAGGGAAGAATACATTTCGACTTGTATCATGTAATAACAAGGACAATAAATCTCCCAACATACACACTAGAGGCTGTATATGAAGCAATTTTTGGAAAGCCAAAGGAGAAGGTATACGCCGACGAGATAGCAAAAGCCTGGGAAAGTGGAGAGAACCTTGAGAGAGTTGCCAAATACTCGATGGAAGATGCAAAGGCAACTTATGAACTCGGGAAAGAATTCCTTCCAATGGAAATTCAGCTTTCAAGATTAGTTGGACAACCTTTATGGGATGTTTCAAGGTCAAGCACAGGGAACCTTGTAGAGTGGTTCTTACTTAGGAAAGCCTACGAAAGAAACGAAGTAGCTCCAAACAAGCCAAGTGAAGAGGAGTATCAAAGAAGGCTCAGGGAGAGCTACACAGGTGGATTCGTTAAAGAGCCAGAAAAGGGGTTGTGGGAAAACATAGTATACCTAGATTTTAGAGCCCTATATCCCTCGATTATAATTACCCACAATGTTTCTCCCGATACTCTAAATCTTGAGGGATGCAAGAACTATGATATCGCTCCTCAAGTAGGCCACAAGTTCTGCAAGGACATCCCTGGTTTTATACCAAGTCTCTTGGGACATTTGTTAGAGGAAAGACAAAAGATTAAGACAAAAATGAAGGAAACTCAAGATCCTATAGAAAAAATACTCCTTGACTATAGACAAAAAGCGATAAAACTCTTAGCAAATTCTTTCTACGGATATTATGGCTATGCAAAAGCAAGATGGTACTGTAAGGAGTGTGCTGAGAGCGTTACTGCCTGGGGAAGAAAGTACATCGAGTTAGTATGGAAGGAGCTCGAAGAAAAGTTTGGATTTAAAGTCCTCTACATTGACACTGATGGTCTCTATGCAACTATCCCAGGAGGAGAAAGTGAGGAAATAAAGAAAAAGGCTCTAGAATTTGTAAAATACATAAATTCAAAGCTCCCTGGACTGCTAGAGCTTGAATATGAAGGGTTTTATAAGAGGGGATTCTTCGTTACGAAGAAGAGGTATGCAGTAATAGATGAAGAAGGAAAAGTCATTACTCGTGGTTTAGAGATAGTTAGGAGAGATTGGAGTGAAATTGCAAAAGAAACTCAAGCTAGAGTTTTGGAGACAATACTAAAACACGGAGATGTTGAAGAAGCTGTGAGAATAGTAAAAGAAGTAATACAAAAGCTTGCCAATTATGAAATTCCACCAGAGAAGCTCGCAATATATGAGCAGATAACAAGACCATTACATGAGTATAAGGCGATAGGTCCTCACGTAGCTGTTGCAAAGAAACTAGCTGCTAAAGGAGTTAAAATAAAGCCAGGAATGGTAATTGGATACATAGTACTTAGAGGCGATGGTCCAATTAGCAATAGGGCAATTCTAGCTGAGGAATACGATCCCAAAAAGCACAAGTATGACGCAGAATATTACATTGAGAACCAGGTTCTTCCAGCGGTACTTAGGATATTGGAGGGATTTGGATACAGAAAGGAAGACCTCAGATACCAAAAGACAAGACAAGTCGGCCTAACTTCCTGGCTTAACATTAAAAAATCCGGTACCGGCGGTGGCGGTGCAACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGTGGTGCGGTAAGCGAAAAGGACGCGCCGAAGGAGCTGCTGCAGATGCTGGAGAAGCAGAAAAAGTGASEQ ID NO: 8 The amino acid sequence of the Pfu- Sso7d fusion proteinMILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWESGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENIVYLDFRALYPSIIITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDIPGFIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYIDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKSGTGGGGATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQMLEKQKKSEQ ID NO: 9 The DNA sequence encoding the Sac7d- ΔTaq fusion proteinATGATTACGAATTCGACGGTGAAGGTAAAGTTCAAGTATAAGGGTGAAGAGAAAGAAGTAGACACTTCAAAGATAAAGAAGGTTTGGAGAGTAGGCAAAATGGTGTCCTTTACCTATGACGACAATGGTAAGACAGGTAGAGGAGCTGTAAGCGAGAAAGATGCTCCAAAAGAATTATTAGACATGTTAGCAAGAGCAGAAAGAGAGAAGAAAGGCGGCGGTGTCACTAGTCCCAAGGCCCTGGAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGTGGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCCCCGAGGGGGTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTGGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCGCCCGCCTCGAGGCCGGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCCGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAACATCCCCGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCGAGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGATAGAGCTCAGGGTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTCTTCCAGGAGGGGCGGGACATCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGGAGGCCGTGGACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTTCGGGGTCCTCTACGGCATGTCGGCCCACCGCCTCTCCCAGGAGCTAGCCATCCCTTACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAGGCCCGGGTGAAGAGCGTGCGGGAGGCGGCCGAGCGCATGGCCTTCAACATGCCCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGGCTATGGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCCCGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGGGCATTGATGGCCGCGGCGGAGGCGGGCATCATCATCATCATCA TTAASEQ ID NO: 10 The amino acid sequence of the Sac7d-ΔTaq fusion proteinMITNSTVKVKFKYKGEEKEVDTSKIKKVWRVGKMVSFTYDDNGKTGRGAVSEKDAPKELLDMLARAEREKKGGGVTSPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEGIDGRGGGGHHHHHHSEQ ID NO: 11 The DNA sequence encoding the PL- ΔTaq fusion proteinATGATTACGAATTCGAAGAAAAAGAAAAAGAAAAAGCGTAAGAAACGCAAAAAGAAAAAGAAAGGCGGCGGTGTCACTAGTGGCGCAACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGTGGTGCGGTAAGCGAAAAGGACGCGCCGAAGGAGCTGCTGCAGATGCTGGAGAAGCAGAAAAAGGGCGGCGGTGTCACCAGTCCCAAGGCCCTGGAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGTGGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCCCCGAGGGGGTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTGGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCGCCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCCGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAACATCCCCGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCGAGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGATAGAGCTCAGGGTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTCTTCCAGGAGGGGCGGGACATCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGGAGGCCGTGGACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTTCGGGGTCCTCTACGGCATGTCGGCCCACCGCCTCTCCCAGGAGCTAGCCATCCCTTACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAGGCCCGGGTGAAGAGCGTGCGGGAGGCGGCCGAGCGCATGGCCTTCAACATGCCCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGGCTATGGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCCCGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGGGCATTGATGGCCGCGGCGGAGGCGGGCATCAT CATCATCATCATTAASEQ ID NO: 12 The amino acid sequence of PL-ΔTaq fusion proteinMITNSKKKKKKKRKKRKKKKKGGGVTSGATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQMLEKQKKGGGVTSPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEGIDGRGGGGHH HHHHSEQ ID NO: 13 PRIMER L71F 5′-CCTGCTCTGCCGCTTCACGC-3′SEQ ID NO: 14 PRIMER L71R 5′-GCACAGCGGCTGGCTGAGGA-3′SEQ ID NO: 15 PRIMER L18015F 5′-TGACGGAGGATAACGCCAGCAG-3′SEQ ID NO: 16 PRIMER L23474R 5′-GAAAGACGA TGGGTCGCTAATACGC-3′SEQ ID NO: 17 PRIMER L18015F 5′-TGACGGAGGATAAC GCCAGCAG-3′SEQ ID NO: 18 PRIMER L29930R 5′-GGGGTTGGAGGTCAATGGGTTC-3′SEQ ID NO: 19 PRIMER L30350F 5′-CCTGCTCTGCCGCTTCACGC-3′SEQ ID NO: 20 PRIMER L35121R 5′-CACATGGTACAGCAAGCCTGGC-3′SEQ ID NO: 21 PRIMER L2089F 5′-CCCGTATCTGCTGGGA TACTGGC-3SEQ ID NO: 22 PRIMER L7112R 5′-CAGCGGTGCTGACTGAATCATGG-3SEQ ID NO: 23 PRIMER L30350F 5′-CCTGCCTGCCGCTTCACGC-3′SEQ ID NO: 24 PRIMER L40547R 5′-CCAATACCCGTTTCA TCGCGGC-3′SEQ ID NO: 25 PRIMER H-Amelo-Y 5′-CCACCTCATCCTGG GCACC-3′SEQ ID NO: 26 PRIMER H-Amelo-YR 5′-GCTTGAGGCCAACCATCAGAGC-3′SEQ ID NO: 27 Human beta-globin primer 536F 5′-GGTTGGCCAATCTACTCCCAGG-3′SEQ ID NO: 28 Human beta-globin primer 536R 5′-GCTCACTCAGTGTGGCAAAG-3′SEQ ID NO: 29 Human beta-globin primer 1408R5′-GATTAGCAAAAGGGCCTAGCTTGG-3′SEQ ID NO: 30 The DNA sequence encoding the Sso7d- ΔTaq(Y) proteinATGATTACGAATTCGAGCGCAACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGTGGTGCGGTAAGCGAAAAGGACGCGCCGAAGGAGCTGCTGCAGATGCTGGAGAAGCAGAAAAAGGGCGGCGGTGTCACTAGTCCCAAGGCcCTGGAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGTGGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCCCCGAGGGGGTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTGGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCGCCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCCGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAACATCCCCGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCGAGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGATAGAGCTCAGGGTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTCTTCCAGGAGGGGCGGGACATCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGGAGGCCGTGGACCCCCTGATGCGCCGGGCGGCCAAGACCATC AAC TACGGGGTCCTCTACGGCATGTCGGCCCACCGCCTCTCCCAGGAGCTAGCCATCCCTTACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAGGCCCGGGTGAAGAGCGTGCGGGAGGCGGCCGAGCGCATGGCCTTCAACATGCCCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGGCTATGGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCCCGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGGGCATTGATGGCCGCGGCGGAGGCGGGCATCATCATCATCATCATTAASEQ ID NO: 31 The amino acid sequence of Sso7d- ΔTaq(Y) proteinMITNSSATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQMLEKQKKGGGVTSPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTI N YGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEGIDGRGGGGHHHHHHSEQ ID NO: 32 The DNA sequence encoding the Sso7d- ΔTaq (E5)(Y) proteinATGATTACGAATTCGAGCGCAACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGTGGTGCGGTAAGCGAAAAGGACGCGCCGAAGGAGCTGCTGCAGATGCTGGAGAAGCAGAAAAAGGGCGGCGGTGTCACTAGTGGGATGCTGCCCCTCTTTGAGCCCAAGGGCCGGGTCCTCCTGGTGGACGGCCACCACCTGGCCTACCGCACCTTCCACGCCCTGAAGGGCCTCACCACCAGCCGGGGGGAGCCGGTGCAGGCGGTCTACGGCTTCGCCAAGAGCCTCCTCAAGGCCCTCAAGGAGGACGGGGACGCGGTGATCGTGGTCTTTGACGCCAAGGCCCCCTCCTTCCCCCACGAGGCCTACGGGGGGCACAAGGCGGGCCGGGCCCCCACGCCAGAGGACTTTCCCCGGCAACTCGCCCTCATCAAGGAGCTGGTGGACCTCCTGGGGCTGGCGCGCCTCGAGGTCCCGGGCTACGAGGCGGACGACGTCCTGGCCAGCCTGGCCAAGAAGGCGGAAAAGGAGGGCTACGAGGTCCGCATCCTCACCGCCGACAAAGACCTTTACCAGCTCCTTTCCGACCGCATCCACGTCCTCCACCCCGAGGGGTACCTCATCACCCCGGCCTGGCTTTGGGAAAAGTACGGCCTGAGGCCCGACCAGTGGGCCGACTACCGGGCCCTGACCGGGGACGAGTCCGACAACCTTCCCGGGGTCAAGGGCATCGGGGAGAAGACGGCGAGGAAGCTTCTGGAGGAGTGGGGGAGCCTGGAAGCCCTCCTCAAGAACCTGGACCGGCTGAAGCCCGCCATCCGGGAGAAGATCCTGGCCCACATGGACGATCTGAAGCTCTCCTGGGACCTGGCCAAGGTGCGCACCGACCTGCCCCTGGAGGTGGACTTCGCCAAAAGGCGGGAGCCCGACCGGGAGAGGCTTAGGGCCTTTCTGGAGAGGCTTGAGTTTGGCAGCCTCCTCCACGAGTTCGGCCTTCTGGAAAGCCCCAAGGCcCTGGAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGTGGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCCCCGAGGGGGTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTGGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCGCCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCCGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAACATCCCCGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCGAGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGATAGAGCTCAGGGTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTCTTCCAGGAGGGGCGGGACATCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGGAGGCCGTGGACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTACGGGGTCCTCTACGGCATGTCGGCCCACCGCCTCTCCCAGGAGCTAGCCATCCCTTACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAGGCCCGGGTGAAGAGCGTGCGGGAGGCGGCCGAGCGCATGGCCTTCAACATGCCCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGGCTATGGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCCCGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGGGCATTGATGGCCGCGGCGGAGGCGGGCATCATCA TCATCATCATTAASEQ ID NO: 33 The amino acid sequence of Sso7d- ΔTaq (E5)(Y) proteinMITNSSATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQMLEKQKKGGGVTSGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFPHEAYGGHKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINYGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDW LSAKEGIDGRGGGGHHHHHH

1. A method of sequencing a target nucleic acid in an aqueous solutionusing an improved DNA polymerase, the method comprising: (a) contactingthe target nucleic acid with a thermally stable DNA polymerase, whereinthe DNA polymerase is joined to a sequence non-specific double-strandednucleic acid binding domain that comprises at least 75% amino acidsequence identity to the Sso7d amino acid sequence set forth in SEQ IDNO:2, and enhances the processivity of the DNA polymerase compared to anidentical DNA polymerase not having the sequence non-specificdouble-stranded nucleic acid binding domain fused to it, and wherein thesolution is of a composition that permits the sequence non-specificdouble-stranded nucleic acid binding domain to bind to the targetnucleic acid and the polymerase domain to extend a primer that ishybridized to the target nucleic acid sequence; (b) incubating thesolution under conditions in which the primer is extended by the DNApolymerase; and (c) identifying the nucleotides that are incorporatedinto the synthesized nucleic acid strand upon the template-dependentextension of the primer by the DNA polymerase, thereby sequencing thenucleic acid.
 2. The method of claim 1, wherein the thermally stable DNApolymerase has a ΔTaq polymerase domain.
 3. The method of claim 1,wherein the sequence non-specific double-stranded nucleic acid bindingdomain comprises at least 90% amino acid sequence identity to the Sso7damino acid sequence set forth in SEQ ID NO:2.
 4. The method of claim 1,wherein the sequence non-specific double-stranded nucleic acid bindingdomain comprises Sso7d (SEQ ID NO:2) or Sac7d (amino acids 7-71 of SEQID NO:10).
 5. A method of sequencing a target nucleic acid in an aqueoussolution using an improved DNA polymerase, the method comprising: (a)contacting the target nucleic acid with a thermally stable DNApolymerase, wherein the DNA polymerase is joined to a sequencenon-specific double-stranded nucleic acid binding domain thatspecifically binds to polyclonal antibodies generated against eitherSso7d (SEQ ID NO:2) or Sac7d (amino acids 7-71 of SEQ ID NO:10), andenhances the processivity of the DNA polymerase compared to an identicalDNA polymerase not having the sequence non-specific double-strandednucleic acid binding domain fused to it, and wherein the solution is ofa composition that permits the sequence non-specific double-strandednucleic acid binding domain to bind to the target nucleic acid and thepolymerase domain to extend a primer that is hybridized to the targetnucleic acid sequence; (b) incubating the solution under conditions inwhich the primer is extended by the DNA polymerase; and (c) identifyingthe nucleotides that are incorporated into the synthesized nucleic acidstrand upon the template-dependent extension of the primer by the DNApolymerase, thereby sequencing the nucleic acid.
 6. The method of claim5, wherein the thermally stable DNA polymerase has a ΔTaq polymerasedomain.
 7. A method of performing a quantitative real-time polymerasechain reaction on a target nucleic acid present in a solution thatcomprises a DNA-binding fluorescent dye, the method comprising: (a)contacting the target nucleic acid with a thermally stable DNApolymerase, wherein the DNA polymerase is joined to a sequencenon-specific double-stranded nucleic acid binding domain thatspecifically binds to polyclonal antibodies generated against eitherSso7d (SEQ ID NO:2) or Sac7d (amino acids 7-71 of SEQ ID NO:10) andenhances the processivity of the DNA polymerase compared to an identicalDNA polymerase not having the sequence non-specific double-strandednucleic-acid binding domain fused to it, wherein the solution comprisesthe DNA-binding fluorescent dye, which exhibits altered fluorescenceemissions when the dye is bound to double-stranded DNA, and is of acomposition that permits the sequence non-specific double-strandednucleic acid binding domain to bind to the target nucleic acid and thepolymerase domain to extend a primer that is hybridized to the targetnucleic acid sequence; (b) incubating the solution under conditions inwhich the primer is extended by the DNA polymerase, (c) exposing thesolution to a suitable excitation light and measuring fluorescenceemission from the DNA-binding fluorescent dye and; (d) performing atleast one additional cycle of amplification comprising steps (a) to (c).8. The method of claim 7, wherein the thermally stable DNA polymerasehas a ΔTaq polymerase domain.
 9. The method of claim 7, wherein thethermally stable DNA polymerase has a Pyrococcus polymerase domain. 10.The method of claim 7, wherein the dye is SYBR Green I.